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Transmural inflammation by interferon--producing T cells correlates with outward vascular remodeling and intimal expansion of ascending thoracic aortic aneurysms

Paul C. Y. Tang, Alexander O. Yakimov, Michael A. Teesdale, Michael A. Coady^*,, Alan Dardik^*,, John A. Elefteriades and George Tellides^*,,1

^* Interdepartmental Program in Vascular Biology and Transplantation, and
Department of Surgery, Yale University School of Medicine, New Haven, Connecticut, USA

¹Correspondence: 295 Congress Ave., Boyer Center for Molecular Medicine 454, New Haven, CT 06510, USA. E-mail: george.tellides{at}yale.edu

SPECIFIC AIMS

Recent clinical and experimental observations in abdominal aortic aneurysms have led to the emerging dogma that intimal hyperplasia and luminal stenosis occur in arterial disease characterized by Th1 immune responses, while arterial enlargement and luminal ectasia result from Th2-dominant immune responses. Because of the limited data available on inflammatory disease of the proximal aorta, we examined whether the cytokine polarization of infiltrating T cells was associated with changes of vessel external diameter and intimal thickness in specimens of non-aneurysmal and aneurysmal ascending thoracic aortas.

PRINCIPAL FINDINGS

1. Transmural leukocytic infiltration characterizes a subgroup of ascending thoracic aortic aneurysms
We counted CD45⁺ (pan-leukocyte marker) cells in aneurysmal and nondilated aortas to assess for the degree of vascular inflammation. All of the aortic specimens had resident leukocytes. Aneurysms had greater medial infiltration than non-aneurysmal aortas (31.1±8.3 vs. 9.0±1.3 CD45⁺ cells/HPF, P<0.01), but there was no significant difference in the density of intimal and adventitial leukocytes (34.6±6.0 vs. 24.6±3.0 and 204.8±38.6 vs. 140.0±26.0 CD45⁺ cells/HPF, respectively). Further analysis showed that disparities in the pattern of aortic inflammation were greatest in the inner half of the media (Fig. 1 A). Only a subgroup (16 of 29) of ascending thoracic aortic aneurysms contained CD45⁺ cells within the inner media; these specimens with transmural mononuclear cellular infiltration are referred to as "infiltrated" aneurysms. The ascending thoracic aortic aneurysms without inflammation of the inner media (13 of 29) are referred to as "bland" aneurysms. None of the non-aneurysmal aortas contained leukocytes within the inner media. The infiltrated aneurysms had a greater density of CD45⁺ cells in every compartment of the vascular wall, whereas there was no difference in the frequency of infiltrating leukocytes between the bland aneurysms and the non-aneurysmal aortas in any arterial layer.

View larger version (31K): [in this window] [in a new window]   Figure 1. A subgroup of aneurysms is characterized by infiltration of the inner media with type I cytokine-producing cells. CD45+ leukocytes (A), CD4++CD8+ T cells (B), CD20+ B cells (C), CD68+ macrophages (D), and transcripts for cytokines (E), chemokine receptors (F), CXCR3 ligands (G), and CCR5 ligands (H) were determined in non-aneurysmal aortas (open bars), bland aneurysms (hatched bars), and infiltrated aneurysms (filled bars). Data are means ±SE. *P< 0.05 vs. bland aneurysms and non-aneurysmal aortas; P < 0.05 vs. non-aneurysmal aortas (ANOVA).

Additional immunostaining was performed to characterize the phenotype of the infiltrating leukocytes. There was a greater number of CD4⁺ and CD8⁺ T cells in the media and adventitia of infiltrated aneurysms compared with non-aneurysmal aortas (Fig. 1B ). Relatively large numbers of CD20⁺ B cells were found in the adventitia of infiltrated aneurysms (Fig. 1C ). CD68⁺ macrophages were the predominant leukocyte cell type in the aortic intima and media, but not in the adventitia of aneurysms, where they were outnumbered by lymphocytes (Fig. 1D ). The sum of the leukocyte subsets that were examined was remarkably similar to the density of CD45⁺ cells, suggesting the great majority of infiltrating immunocytes had been identified.

2. Th1-type immune responses predominate in ascending thoracic aortic aneurysms
We further characterized the vascular infiltrating cells by their expression of cytokine and chemokine receptor to determine if a particular T lymphocyte subset was associated with aortic aneurysm formation. Transcript expression was quantified by real-time RT-PCR. There was greater expression of Th1 cytokine transcripts in aneurysms compared with non-aneurysmal aortas (0.21±0.06 vs. 0.03±0.01 IFN-/GAPDH RNA x10^–3, P<0.05), undetectable Th2 cytokine (IL-4, IL-5, and IL-13) transcripts in all specimens, and a trend to greater expression of Th3 cytokine transcripts in non-aneurysmal aortas compared with aneurysms (6.31±1.98 vs. 2.45±0.84 IL-10/GAPDH RNA x10^–3, P=0.053). Additional analysis showed that the increased expression of IFN- transcripts was largely accounted for by the infiltrated subgroup of aortic aneurysms (Fig. 1E ). There was also a greater expression of CXCR3 (chemokine receptor expressed primarily on IFN--producing T cells), but not CCR5 (expressed on a subset of Th1 cells), mRNA in the infiltrated aneurysms compared with the bland aneurysms or non-aneurysmal aortas (Fig. 1F ). The production of IFN- by infiltrating leukocytes was confirmed at the protein level and two-color immunostaining colocalized the cytokine to CD4⁺ and CD8⁺ T cells. Expression of CXCR3 protein was also readily detected in infiltrated aneurysms, but not non-aneurysmal aortas.

These findings raised the possibility that IFN--producing cells are selectively recruited to the vessel wall of infiltrated aneurysms. Since Th1 cells preferentially respond to CXCR3 binding chemokines, we examined their expression in aortic tissue. Both IP-10 and Mig transcripts were up-regulated in aneurysms with transmural inflammation (Fig. 1G ). Moreover, there was greater expression of IP-10 and Mig protein in infiltrated aneurysms than non-aneurysmal aortas as detected by immunohistochemistry. Transcripts for the CCR5 ligands RANTES and MIP-1ß were not specific for infiltrated aneurysms (Fig. 1H ). The presence of IFN--inducible chemokines, such as IP-10 and Mig, implies that IFN- is active on cells of the vessel wall and may play a role in recruiting additional Th1 cells to the infiltrated aneurysms. Together, the data show that cellular infiltrates of aortic aneurysms, particularly those with transmural inflammation, are characterized by IFN--secreting T lymphocytes.

3. Transmural inflammation and IFN- production are associated with increased aortic external diameter and intimal thickening
We investigated whether the presence of a transmural Th1-type lymphocytic infiltrate was associated with morphological or cellular changes of the aorta. The infiltrated aneurysms were significantly larger than the bland aneurysms (Fig. 2 A). This was not due to anthropomorphic differences between the groups, as their predicted external diameters were almost identical. The vessel wall and adventitia thickness were similar for all the aortic specimens (Fig. 2B ). In contrast, the intima was thicker and the media was thinner in the infiltrated aneurysms (Fig. 2C ).

View larger version (37K): [in this window] [in a new window]   Figure 2. Infiltrated aneurysms have increased size and intimal thickness, but preserved VSMCs. Actual and predicted external diameter (A), wall thickness (B, C), SM -actin+ cells (D), SM -actin immunostaining (E), and SM -actin transcript expression (F) were determined in non-aneurysmal aortas (open bars), bland aneurysms (hatched bars), and infiltrated aneurysms (filled bars). Data are means ± SE. *P< 0.05 vs. bland or infiltrated aneurysms and non-aneurysmal aortas (ANOVA).

Since the media may thin secondary to stretching from vascular dilatation alone, we calculated the medial area and found it similar for both infiltrated (196.4±16.1 mm²) and bland (210.3±10.5 mm²) aneurysms and significantly greater than that of the non-aneurysmal aortas (126.4±4.8 mm²; P<0.001). In keeping with an absence of medial atrophy, the density of VSMCs was preserved in the media of ascending thoracic aortic aneurysms (Fig. 2D ). Moreover, the expression of SM -actin (VSMC marker) protein and transcripts was not different between infiltrated aneurysms and noninflamed aortas (Fig. 2E, F ).

There were significant correlations between the expression of IFN- transcripts vs. external aortic diameter (r=0.30, P<0.05) and intimal thickness (r=0.33, P<0.05), but not with SM -actin⁺ cells (r0.03), SM -actin protein (r=0.07), and SM -actin mRNA (r0.06). These results establish that leukocytic infiltration of the inner media and Th1-type immune responses correlate with both aneurysm size and intimal hyperplasia, but not with loss of VSMCs.

4. Transmural inflammation and IFN- production are associated with matrix degradation
Finally, we determined the expression of matrix proteins by quantitative image analysis of sirius red, alcian blue, and elastin staining, which largely label collagen, sulfated forms of ground substance, and elastin, respectively. Collagen staining was decreased in the inner media of infiltrated aneurysms vs. non-aneurysmal aortas (47.7±4.3 vs. 68.7±3.0%, P<0.001). The extent of staining for ground substance and elastin was less in the inner media of infiltrated aneurysms compared with both bland aneurysms and non-aneurysmal aortas (53.8±7.6 vs. 82.7±3.3 and 81.6±2.5%, P<0.001; 14.7±2.9 vs. 43.1±5.1 and 50.4±3.1%, P<0.001, respectively). Elastin fragmentation was also more extensive in infiltrated aneurysms, but no different between bland aneurysms and control aortas (3.5±0.2 vs. 0.5±0.1 and 0.5±0.1 grade, P<0.001).

IFN- transcript expression had significant correlations with inner media sirius red (r0.48, P<0.001) and alcian blue (r0.42, P<0.01) staining, and elastin fragmentation (r=0.44, P<0.01). The data demonstrate that IFN--related outward vascular remodeling is characterized by matrix degradation.

CONCLUSIONS AND SIGNIFICANCE

The results of the present study support a number of conclusions regarding the inflammation and remodeling of ascending thoracic aortic aneurysms (Fig. 3 ). First, leukocytes are present in the intima, outer media, and adventitia of both nondilated and aneurysmal proximal thoracic aortas. Leukocytic infiltration of the inner media is a simple pathological marker for vascular inflammation and is found in approximately half of ascending thoracic aortic aneurysms. Second, aortic aneurysms, particularly those with transmural inflammation, are distinguished by Th1-type immune responses with activated CD4⁺ and CD8⁺ T lymphocytes producing IFN- in situ. Recruitment of CXCR3⁺ T cells is associated with secretion of the IFN--inducible chemokines IP-10 and Mig. Third, inflammation of the inner media and IFN- production correlate with both outward vascular remodeling and intimal expansion of the ascending thoracic aorta. The remodeling of infiltrated ascending thoracic aortic aneurysms is characterized by medial matrix degradation, but preserved VSMC density. A corollary is that bland aneurysms do not exhibit overt evidence of matrix loss or fragmentation of elastin as compared with non-aneurysmal aortas.

View larger version (22K): [in this window] [in a new window]   Figure 3. Transmural infiltration by IFN--producing T cells is associated with outward vascular remodeling and intimal expansion of the ascending thoracic aorta. Vessel diameter and wall thickness are drawn to scale based on data means. The intima (I), media (M), and adventitia (A) are drawn at 10x magnification and infiltrating leukocytes are represented as shaded circles. Inflammation of the inner media is a simple pathological marker that differentiates infiltrated from bland aneurysms as compared with non-aneurysmal aortas. Aneurysms infiltrated by Th1-type T cells have a larger external diameter and a thicker intima than bland aneurysms.

Our novel findings in ascending thoracic aortic aneurysms of the predominance of Th1-type immune responses and the absence of medial atrophy or loss of VSMCs differ from the current understanding of vascular inflammation and remodeling in aneurysm pathogenesis based on studies of abdominal aortic aneurysms. Our observations of the ubiquitous presence of intimal and adventitial leukocytic infiltrates in all aortic specimens and the pathological significance of inner media inflammation reinforce the concepts of vascular-associated lymphoid tissue and medial immunoprivilege. We have previously shown in experimental models using immunodeficient mouse hosts that IFN- is sufficient and necessary for intimal expansion and outward vascular remodeling of human arteries. The present study demonstrates a positive correlation between transmural inflammation by IFN--producing T cells and increasing vessel size and intimal hyperplasia in clinical specimens of ascending thoracic aortic aneurysms.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3671fje;

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This Article Full Text (PDF) All Versions of this Article: 19/11/1528 05-3671fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tang, P. C. Y. Articles by Tellides, G. Search for Related Content PubMed PubMed Citation Articles by Tang, P. C. Y. Articles by Tellides, G.